In the last decade, genomes of more than 200 bacteria (including more than 100 pathogens) have become available, opening tremendous opportunities. Comparative and functional genomics strongly impact our ability to explore connections between metabolism and virulence, host-pathogen interactions, and other important aspects of infectious diseases. At the same time, the avalanche of sequence data has created a new annotation challenge. In spite of recent progress, a large fraction of genes in most of the available genomes remains incorrectly or imprecisely annotated. One of the motivations of the proposed cross-disciplinary research project is to establish an integrated approach to generate reliable and consistent genomic annotations accurately projected over the collection of sequenced genomes. Our approach combines an optimized experimental validation strategy with bioinformatics techniques, reconstruction of metabolic subsystems, and genome context analysis. In the proposed study, we will apply this approach to develop a comprehensive genomic analysis of three subsystems: (1) Coenzyme A, (2) NAD and NADP, and (3) FMN and FAD metabolism in a variety of bacterial pathogens and commensals. We will validate selected gene assignments, elementary pathways, and their combinations (functional variants) by a variety of biochemical and genetic techniques. The coenzymes chosen for our study are involved in hundreds of biochemical reactions and regulatory processes, and are essential in all forms of cellular life. Our previous studies implicated several key enzymes involved in their biosynthesis as potential anti-infective drug targets. Accurate global mapping of the respective subsystems will provide us with a functional context for such targets and will contribute to the fundamental understanding of core metabolism in pathogens.